Anti-Inflammatory Effects of Flavonoids in Citrus Jabara Fruit Peels

Trends in Immunotherapy (2020) Volume 4 Issue 1. doi: 10.24294/ti.v4.i1.844

Original Article Anti-inflammatory effects of flavonoids in Citrus jabara fruit peels

Seisho Azuma1,2§, Yoshinobu Murakami1§*, Eiko Azuma2, Kimiye Baba1, Masahiko Taniguchi1 1 Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan 2 Jabara Laboratory, 4-7-18 Matsue Kita, Wakayama 640-8425, Japan §: Both authors contributed equally to this work.

ABSTRACT Currently, about half of people in Japan suffer from allergic diseases. Thus, Citrus jabara fruits have been paid attention as one of quite effective anti-allergic functional foods. C. jabara is an endemic species originally grown only in Kitayama village, Wakayama prefecture in Japan. Although genetic characterization and diversity of various Citrus fruits including C. jabara were researched, but there is room for the study on flavonoids characteristics in C. jabara fruit. For the alleviation of allergic symptom, anti-inflammatory effects are also important. In this study, characteristics of flavonoids in C. jabara fruit peels, and the anti-inflammatory effects of these purified flavonoids were investigated. Our results revealed that C. jabara is a unique Citrus that almost all of flavonoids in fruit peels was narirutin. There was no Citrus species with a flavanone glycosides content ratio like C. jabara. Although anti-inflammatory effects of narirutin was weak, but its aglycone naringenin exhibited following inhibitory effects: nitric oxide synthesis (IC50 = 105 μM), nitric oxide synthase induction, Interleukin-6 synthesis (IC50 = 65 μM), and inducible soluble epoxide hydrolase activity

(IC50 = 267 μM). Since narirutin is deglycosylated to naringenin that is then absorbed by colonocytes, it is considered that narirutin exists like a prodrug and its aglycone naringenin works as an active form of anti-inflammatory effect in a living body at oral ingestion of C. jabara fruit peels. Keywords: Citrus jabara; anti-inflammatory effects; flavonoids; narirutin; naringenin

ARTICLE INFO Received: March 6, 2020 Introduction Accepted: April 1, 2020 Available online: April 15, 2020 Currently, about half of people in Japan suffer from allergic diseases such as pollen allergy, perennial rhinitis, and atopic dermatitis. So, it is figuratively *CORRESPONDING AUTHOR said that allergic diseases are the current national disease in Japan. Under such Yoshinobu Murakami, 4-20-1 condition, the functional foods to alleviate allergic symptoms, such as Citrus Nasahara, Takatsuki, Osaka 569-1094, [1,2] Japan; [email protected] unshiu unripe fruit, were focused .

CITATION In recent years, Citrus jabara fruits has also been paid attention as one of Azuma S, Murakami Y, Azuma E, et quite effective anti-allergic functional foods. C. jabara is an endemic species al. Anti-inflammatory effects of flavonoids in Citrus jabara fruit peels. originally grown only in Kitayama village, Wakayama prefecture in Japan, Trends Immunother 2020; 4(1): 5-14. and is estimated as a natural hybrid species of Citrus junos, Citrus reticulata, doi: 10.24294/ti.v4.i1.844 and Citrus kinokuni[3]. C. jabara was recognized as a new variety of citrus by Dr. Tanaka in 1971, and then registered as a new nursery plant in 1979 by Mr. COPyRIGHT Copyright © 2020 by author(s) and Fukuda (Japan the plant variety protection NO. 10). Concerning with anti- EnPress Publisher LLC. This work is allergic activities of C. jabara fruits, the anti-degranulation activity[3], the licensed under the Creative Commons improving effect of its juice on symptoms and quality of life in patients with Attribution-NonCommercial 4.0 [4] International License (CC BY-NC 4.0). Japanese cedar pollinosis , and the suppressive effect of its extracts on mucin http://creativecommons.org/licenses/ production in human lung epithelial cells[5] were reported. Although genetic by/4.0/ characterization and diversity of various citrus fruits including C. jabara were researched [6–8], but there is room for the study on flavonoids characteristics in C. jabara fruit. For the alleviation of allergic symptom, both anti-allergic effect and anti-inflammatory effects are important. Thus, in this study, we investigated the component analysis of flavonoids in C. jabara fruit peels and the anti-inflammatory effects of these isolated flavonoids.

5 Anti-inflammatory effects of flavonoids in Citrus jabara fruit peels

Materials and Methods (FLV6, 2.9 mg), retusin (5-hydroxy-3, 3’, 4’, 7-tetramethoxyflavone) (FLV7, 1.9 mg), Extract and isolation nobiletin (3’, 4’, 5, 6, 7, 8-hexamethoxyflavone) (FLV8, 1.9 mg), three flavanones: 3’, 4’, 5, 6, 7, C. jabara fruits were harvested in Kitayama 8-hexamethoxyflavanone (FNN2, 32.1 mg), 3’, village, Wakayama prefecture in Japan and on 3 4’, 5, 6, 7-pentamethoxyflavanone (FNN3, 6.7 November 2010. The residue of C. jabara fruits by mg), 4’, 5, 6, 7-tetramethoxyflavanone (FNN4, commercial roller juice squeezer was used as C. 1.4 mg), three chalcones: 2’-hydroxy-3, 3’, 4, 4’, jabara fruit peels. After drying, these peels were 5’, 6’-hexamethoxychalcone (CHL1, 31.7 mg), chopped and sealed in aluminum puck until use. 2’-hydroxy-3, 4, 4’, 5’, 6’-pentamethoxychalcone Extract and isolation were carried out in 2010. ( CHL2, 8.2 mg), 2’-hydroxy-4, 4’, 5’, 6’-tetramethoxychalcone (CHL3, 7.8 mg). The The dried of C. jabara fruit peels (1 Kg) EtOAc and acetone residues were filtered to obtain were chopped into small pieces and extracted an amorphous powder (narirutin: FNN1, 17.7 g). with n-hexane (3 L x 5), EtOAc (3 L x 3) and These compounds were determined by comparison acetone (3 L x 3) using a ultrasonic extraction. of physical data and NMR spectral data with The combined n-hexane, EtOAc and acetone [9–13] articles . extracts were concentrated to dryness in vacuo, respectively. The n-hexane residue (11.7 g) was Chemical structures of isolated compounds were subjected to column chromatography on silica shown in Figure 1, and their names and amounts gel and ODS, and eluted successively with a were listed in Table 1. Among these compounds, n-hexane – EtOAc and MeOH – H2O solvent FLV3, 5, 6, 7 and CHL1-3 are the compounds which system to afford fourteen compounds, eight were isolated for the first time from C. jabara fruit flavones: 3, 3’, 4’, 5, 6, 7, 8-heptamethoxyflavone peels[14]. Seven compounds that were obtained more (FLV1, 265.8 mg), natsudaidain (3-hydroxy-3’, than 10 mg (FLV1 - 4, FNN1 - 2, and CHL1) were 4’, 5, 6, 7, 8-hexamethoxyflavone) (FLV2, evaluated in cell-based assays. 130.3 mg), 5-hydroxy-3, 3’, 4’, 6, 7, 8-hexamethoxyflavone (FLV3, 29.3 mg), Materials 3, 3’, 4’, 5, 6, 7-hexamethoxyflavone (FLV4, 14.6 mg), 8-hydroxy-3, 4’, 5, 6, Naringenin was purchased from LKT Laboratory 7-pentamethoxyflavone (FLV5, 7.1 mg), 4’, (St. Paul, MN, USA). Hesperidin, neohesperidin, 5-dihydroxy-3, 3’, 6, 7, 8-pentamethoxyflavone and naringin were purchased from Nacalai Tesque

Figure 1. Structures of isolated flavonoids from dried C. jabara fruit peels

6 Azuma S, et al.

Table 1. Flavonoids isolated from dried C. jabara fruit peels (1 kg).

Compound Amount (mg) Flavones FLV1 3, 3’, 4’, 5, 6, 7, 8-heptamethoxyflavone 265.8 FLV2 natsudaidain (3-hydroxy-3’, 4’, 5, 6, 7, 8-hexamethoxyflavone) 130.3 FLV3 5-hydroxy-3, 3’, 4’, 6, 7, 8-hexamethoxyflavone 29.3 FLV4 3, 3’, 4’, 5, 6, 7-hexamethoxyflavone 14.6 FLV5 8-hydroxy-3, 4’, 5, 6, 7-pentamethoxyflavone 7.1 FLV6 4’,5-dihydroxy-3, 3’, 6, 7, 8-pentamethoxyflavone 2.9 FLV7 retusin (5-hydroxy-3, 3’, 4’, 7-tetramethoxyflavone) 1.9 FLV8 nobiletin (3’, 4’, 5, 6, 7, 8-hexamethoxyflavone) 1.9 Flavanones FNN1 narirutin 17,700.0 FNN2 3’, 4’, 5, 6, 7, 8-hexamethoxyflavanone 32.1 FNN3 3’, 4’, 5, 6, 7-pentamethoxyflavanone 6.7 FNN4 4’, 5, 6, 7-tetramethoxyflavanone 1.4 Chalcones CHL1 2’-hydroxy-3, 3’, 4, 4’, 5’, 6’-hexamethoxychalcone 31.7 CHL2 2’-hydroxy-3, 4, 4’, 5’, 6’-pentamethoxychalcone 8.2 CHL3 2’-hydroxy-4, 4’, 5’, 6’-tetramethoxychalcone 7.8

(Kyoto, Japan). Dulbecco’s modified Eagle’s medium (TNF-α) ware obtained from BioLegend (San Diego, (DMEM) and penicillin-streptomycin mixed solution CA, USA). Human recombinant soluble epoxide (stabilized) were from Nacalai Tesque. Fetal bovine hydrolase (sEH) was purchased from Cayman serum was obtained from Biowest (Nuaillé, France). Chemical Company (Ann Arbor, MI, USA), and its Lipopolysaccharide (LPS) from Escherichia coli O55 substrate, (2S,3S)-trans-3-phenyl-2-oxiranylmethyl- was obtained from Wako Pure Chemical Industries 4-nitrophenyl carbonate (S-NEPC), was from Sigma- (Osaka, Japan). Griess reagent system was obtained Aldrich. Human recombinant chymase was obtained from Promega (Fitchburg, WI, USA). Cell Counting from Sigma-Aldrich, and its substrate, N-Succinyl- Kit-8 was from Dojindo Laboratories (Kumamoto, Ala-His-Pro-Phe-p-nitroanilide, was from Peptide Japan). RIPA buffer and protease inhibitor cocktail Institute (Osaka, Japan). Hyaluronidase from were purchased from Wako Pure Chemical. BCA bovine testes and Compound 48/80 were obtained Protein Assay Kit was obtained from Thermo Fisher from Sigma-Aldrich. Hyaluronic acid sodium salt Scientific (Waltham, MA, USA). Polyvinyliden from Rooster Comb was obtained from Wako Pure fluoride (PVDF) membrane was obtained from Bio- Chemical Industries. Rad (Hercules, CA, USA). Blocking one, a blocking solution for blot membrane, was obtained from HPLC analysis for quantification of Nacalai Tesque. Rabbit antibody to inducible nitric hesperidin, neohesperidin, naringin, and oxide synthase (iNOS) was obtained from Sigma- naringenin Aldrich (St. Louis, MO, USA), and anti-rabbit IgG HRP-linked whole antibody was from GE Healthcare Prior to the sample preparation, dried C. jabara (Buckinghamshire, UK). Monoclonal antibody to fruit peels were powdered. Amounts of hesperidin, neohesperidin, naringin, and naringenin were β-actin HRP conjugate was obtained from Wako Pure [15] Chemical Industries. Amersham ECL Prime Western quantified as reported by Tosa et al. with some Blotting Detection Reagent, chemiluminescent modifications. Briefly, 50 mg of C. jabara powder reaction reagent, was obtained from GE Healthcare. were taken into a 10 mL vial and extracted with 50% Enzyme linked immunosorbent assay (ELISA) kits methanol for 20 min using an ultrasonic extraction. for Interleukin 6 (IL-6) and tumor necrosis factor α Then, extract was passed through a 0.45 μm syringe

7 Anti-inflammatory effects of flavonoids in Citrus jabara fruit peels filter into a glass vial, and 5.0 μL was injected ± 0.121 nM, and that of TNF-α protein was 5.030 into the HPLC. Chromatographic separation was ± 0.774 nM, respectively. The inhibitory ratio was performed on a COSMOSIL 5C18-AR-II column calculated by the following formula: Inhibitory ratio (φ 4.6 x 250 mm) with a following condition: (%) = (1-average of treated group/ average of control temperature, 40 °C; mobile phase, 20% acetonitrile group) x 100. containing 0.8% acetic acid; flow rate, 1.0 mL/min. Quantitative analysis was performed with purchased Western blot analysis standard samples of hesperidin, neohesperidin, RAW 264 cells were seeded in φ 60 mm dishes naringin, and naringenin. Peaks were detected at 6 280 nm. The limits of detection of each flavanone at a density of 6.0 x 10 cells/dish in DMEM with were follows: hesperidin, 40 ng/mL; neohesperidin, 10% FBS at 37 °C for 2 h. Next, cells were cultured 92 ng/mL; naringin, 65 ng/mL; naringenin, with or without flavonoids for 2 h, then, stimulated 10 ng/ml, respectively. The quantification limits of with 1 μg/mL of LPS for an additional 24 h. After each flavanone were follows: hesperidin, 120 ng/mL; treatment, cells were washed and lysed with RIPA neohesperidin, 280 ng/mL; naringin, 200 ng/mL; buffer and centrifuged at 20,600 xg for 30 min at naringenin, 30 ng/mL, respectively. 4 °C. Protein concentrations of supernatants were estimated by BCA protein assay kit, and stored at Cell culture -80˚C until analysis. Equal amounts of protein (2.5 μg) of the cell lysates were separated by sodium dodecyl The murine macrophage-like cell line, RAW sulfate-polyacrylamide gel electrophoresis (SDS- 264 (RCB0535), was provided from the RIKEN PAGE) and transferred to PVDF membranes. The BioResource Center (Ibaraki, Japan). RAW 264 cells membranes were pre-incubated in blocking solution were cultured in DMEM containing 10% FBS, 100 at room temperature for 1 h. For iNOS protein units/mL penicillin, and 100 μg/ mL streptomycin. detection, a membrane was incubated with a Cells were incubated at 37 °C with 5% CO2 in a 1: 2,000 dilution of a primary antibody against iNOS humidified atmosphere. at room temperature for 1 h. The blot was washed with T-PBS (-) and incubated with a 1:4,000 dilution Cell viability assay of HRP-conjugated IgG secondary antibody. In case of β-actin, the transferred membrane was incubated The cytotoxicity of flavonoids on RAW 264 cells with a 1:8,000 dilution of a HRP-conjugated were measured by Cell Counting Kit-8. Briefly, 1.0 x antibody against β-actin at room temperature for 1 h. 105 cells/well of RAW 264 cells were seeded on a 96 The target proteins on the membranes were detected well plate in DMEM with 10% FBS and antibiotics using a chemiluminescent reaction and LAS-3000 with various flavonoids at 37 °C for 24 h. Then, cells Imaging System (Fuji film, Tokyo, Japan). were washed, and added new 200 μL of DMEM with 10% FBS and 10 μL of WST-8 reagent. After further Enzyme inhibitory assay incubation for 2 h, cell viability was determined by measuring the absorbance at a wavelength of 450 sEH activity was measured as reported by Bahl et nm. The cell viability ratio was calculated by the al.[16] with some modifications. Briefly, samples were following formula: Cell viability (%) = (average of incubated at 37 °C for 30 min in a 100 μL volume treated group/average of control group) x 100. containing 0.58 U/mL sEH and 0.4 mM s-NEPC. Hyaluronidase activity was measured by the Measurement of nitric oxide (NO), IL-6, and Morgan-Elson reaction as reported by Ratnasooriya TNF-α production et al.[17] Chymase activity was measured as reported by Tani et al.[18] with some modification. Briefly, RAW 264 cells were seeded in a 96 well plate at 5 samples were incubated at 37 °C for 60 min in a 100 μL a density of 1.0 x 10 cells/well in DMEM with 10% volume containing 8.8 μg/mL chymase and 0.8 mM FBS and antibiotics at 37 °C for 2 h. Next, cells were N-Succinyl-Ala-His-Pro-Phe-p-nitroanilide. These cultured with or without flavonoids for 2 h, then, assays were carried out in 96-well plates and the stimulated with 1 μg/mL of LPS for an additional optical density was measured at 405 nm in case 24 h. After treatment, culture supernatants were of sEH and chymase, and at 585 nm in case of collected and stored -20 °C until assayed. The NO hyaluronidase, respectively. The inhibitory ratio concentration was measured by using Griess reagent was calculated by the following formula: Inhibitory system. Average of LPS-induced NO production ratio (%) = (1-average of increase in absorbance of was 26.5 ± 1.4 μM. The concentration of IL-6 and sample/ average of increase in absorbance of control) TNF-α was measured by using mouse ELISA kits. x 100. Average of LPS-induced IL-6 protein was 1.034

8 Azuma S, et al.

Results were examined until 500 µM. Narirutin up to 500 µM and naringenin up to 400 µM exhibit no toxicity. At Flavonoids in C. jabara fruit peels 500 µM naringenin, cell viability was 85.8 ± 6.0 %. Amounts of flavonoids in C. jabara fruit Flavonoids in C. jabara fruit peels inhibit peels were quantified to clarify the distribution LPS-induced NO production and iNOS characteristics in C. jabara fruit peels. Eight protein generation in RAW 264 cells flavones, 4 flavanones, and 3 chalcones were detected in C. jabara fruit peels, but more than In order to investigate the suppressive effects on 99% of flavonoids were narirutin Table( 1). Typical NO synthesis and iNOS inhibitory activities, RAW flavanone glycosides of Citrus fruit peels, such as 264 cells were cultivated with 1 µg/mL of LPS and hesperidin, neohesperidin, nor naringin, were not 50, 100, and 200 µM of flavonoids for 24 h. NO detected during the isolation and quantification concentrations in the culture supernatants were process of C. jabara fruit peels. Furthermore, quantified by the Griess reaction. In case of FLV4 naringenin, an aglycone of narirutin, was also not at 200 µM, NO was not evaluated because of its detected. low solubility. Expression levels of iNOS protein at 100 µM flavonoids were visualized by immunoblot For confirmation, amounts of hesperidin, analysis. All tested flavonoids, except for narirutin, naringin, neohesperidin, and naringenin in C. jabara exhibited inhibitory effects equally or more than that fruit peels were quantified by HPLC analysis. of L-NAME (Figure 3A). With a few exceptions, Amounts of these flavonoids were below detection dose-dependently inhibitory effects were observed limit. on each tested flavonoid. These flavonoids, except for narirutin, significantly reduced LPS-induced Toxicities of flavonoids in C. jabara fruit peels iNOS protein expression, but did not change β-actin expression (Figure 3B). Prior to investigate the anti-inflammatory effects, toxicities of purified flavonoids in C. jabara fruit Flavonoids in C. jabara fruit peels inhibit peels were examined. Cell viabilities of RAW 264 LPS-induced IL-6 and TNF-α secretion in cells cultivated with 100 and 200 µM flavonoids for RAW 264 cells 24 h were measured with Cell Counting Kit-8. In case of FLV4, toxicity of 200 µM was not evaluated In order to investigate the suppressive effects because of its low solubility. At 100µM, no toxicity on IL-6 and TNF-α secretion, RAW 264 cells were was observed on FLV4, narirutin, and its aglycone cultivated with 1 µg/mL of LPS and 50, 100, and naringenin (Figure 2). Mild toxicity was observed 200 µM of flavonoids for 24 h. IL-6 and TNF-α on FLV1-3 and FNN2. Strong toxicity was observed concentrations in the culture supernatants were on CHL1. Toxicities of narirutin and naringenin quantified by the ELISA kits. In case of FLV4 at 200

Figure 2. Toxicities of flavonoids in C. jabara fruit peels on RAW 264 cells. Flavonoids were tested at concentrations of 100 μM( ■ ) and 200 μM( ■ ). The values express the means ± S.D. of three experiments. NRT, narirutin; NGN, naringenin. Other abbreviations of flavonoids are listed in Table 1. N.D., no data.

9 Anti-inflammatory effects of flavonoids in Citrus jabara fruit peels

µM, IL-6 and TNF-α were not evaluated because (sEH), hyaluronidase and chymase. Dose-dependent of its low solubility. Dose-dependently inhibitory inhibitory effects were observed other than chymase effects were observed on each tested flavonoid. (Figure 5). The inhibitory concentration 50% Among these flavonoids, FNN2, naringenin, and (IC50) values of naringenin on inflammatory related FLV1 inhibited IL-6 secretion strongly, but narirutin factors were follows; NO production was 105 and FLV3 did weakly (Figure 4A). In case of μM, IL-6 secretion was 65 μM, sEH was 267 μM, TNF-α, only FNN2 exhibited strong inhibitory and hyaruronidase was 1,470 μM, respectively. activity (Figure 4B). On the other hand, narirutin did not exhibit significant inhibitory effects in the range of tested Inhibitory effects of narirutin and naringenin concentrations. on inflammatory related factors Discussion Inhibitory effects of narirutin and its aglycone naringenin on following inflammatory related factors In this study, we clarified the characteristics of were examined; secretions of NO, IL-6 and TNF-α flavonoids in C. jabara fruit peels, and investigated in LPS-stimulated RAW 264 cells, and inflammation the anti-inflammatory effects of these purified related enzymes, such as soluble epoxide hydrolase flavonoids. Furthermore, we estimated the most

Figure 3. Flavonoids in C. jabara fruit peels inhibit LPS-induced NO production (A) and iNOS generation (B) in RAW 246 cells. For NO production, flavonoids were tested at concentrations of 50 μM( ■ ), 100 μM( ■ ), and 200 μM( ■ ). For iNOS generation, flavonoids were tested at concentration of 100.The values express the means ± S.D. of three experiments. NRT, narirutin; NGN, naringenin; L-NAME, N-Nitro-L-arginine methyl ester hydrochloride (positive control). Other abbreviations of flavonoids are listed inTable 1. N.D., no data.

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Figure 4. Flavonoids in C. jabara fruit peels inhibit LPS-induced IL-6 (A) and TNF-α (B) in RAW 246 cells. Flavonoids were tested at concentrations of 50 μM( ■ ), 100 μM( ■ ), and 200 μM( ■ ). The values express the means ± S.D. of three experiments. NRT, narirutin; NGN, naringenin; L-NAME. Other abbreviations of flavonoids are listed in Table 1. N.D., no data. effective anti-inflammatory compound in a living limit. There was no Citrus species with a flavanone body. glycosides content ratio like C. jabara[19,20]. This result indicates that C. jabara is a unique citrus. Citrus could be classified by the content ratio of following 4 flavanone glycosides, such as narirutin, Flavonoids in C. jabara fruit peels did not hesperidin, naringin, and neohesperidin, in its exhibit strong toxicity except for CHL1 (Figure fruits[19]. Principal component analysis of Citrus 2). Especially, narirutin and its aglycone naringenin fruits flavonoids indicated that distributions of Citrus were nontoxic. Thus, C. jabara fruit peel is species belonging to different classes were largely in considered as a nontoxic material, because almost all accordance with Tanaka’s classification system[20]. C. of flavonoids are narirutin inC. jabara fruit peels. jabara is recognized as a closely-related species to C. sphaerocarpa, C. sudachi, C. junos, and C. reticulate Anti-inflammatory effects of flavonoids were by genetic characterization and diversity research[6–8]. observed on inflammatory mediators in LPS- As shown in Table 1, almost all of flavonoids in stimulated macrophage-like cells (Figures 3 and 4). Flavonoids are known as suppressors of C. jabara fruit peels was narirutin, but hesperidin, [21,22] naringin, and neohesperidin were below detection nuclear factor κB (NF-κB) pathway . Among

11 Anti-inflammatory effects of flavonoids in Citrus jabara fruit peels

Figure 5. Inhibitory effects of narirutin and naringenin on inflammatory factors. (A)NO, (B)IL-6, (C)TNF-α, (D)sEH, (F)hyaluronidase, and (F)chymase were examined. The values express the means S.D. of three experiments. tested flavonoids, FNN2 was most effective on Thinking about the abundance ratio of flavonoids, NO synthesis, iNOS induction, IL-6 synthesis, and narirutin is considered to contribute to anti- TNF-α synthesis (Figures 3 and 4). These results inflammatory effect at oral ingestion of C. jabara may be effected by the reduction of cell viability, fruit peels. Funaguchi et al. also reported that thus, toxicities of each flavonoid should also be oral administration of narirutin inhibited airway taken into consideration. Although anti-inflammatory inflammation in allergic model mouse[25]. On the other effects of narirutin was weak, but, its aglycone hand, in vitro cell assay, narirutin did not exhibited naringenin exhibited enough inhibitory effects enough anti-inflammatory effects (Figures 2–5) (Figures 3–5). The difference in the inhibitory nor anti-degranulating effects[26]. These conflicting activity of narirutin and naringenin, in other word, results between in vivo and in vitro examinations glycosides and aglycone, may be partially explained may be explained by the metabolism of narirutin in by the difference in cell membrane permeability[23,24]. a living body. Pharmacokinetics of narirutin after In this study, other structure-activity relations of ingestion of orange juice in healthy humans were inhibitory effects of tested flavonoids were not clear. reported by Silveria et al.[27]. The majority of oral For example, Takano-Ishikawa et al. reported that intake-narirutin is considered to reach the colon, C2-C3 double-bond and 4-oxo functional group of and colonic microbiota deglycosylate narirutin to the C-ring in flavonoids are important factors for the naringenin, that is then absorbed by colonocytes[28]. high inhibition activities of prostaglandin E2 (PGE2) Thus, it is considered that narirutin exists like a induction by LPS in rat peritoneal macrophages[23]. In prodrug, and its aglycone naringenin works as an this study, all tested flavonoids have 4-oxo functional active form within the body. group in the C-ring. The C2-C3 double bond of C-ring, in short, flavone (FLV) skeletal structure, Anti-inflammatory effects of naringenin were also was not relevant to the inhibition activities in our observed on inflammation related enzymes, such results (Figures 2–4). FNN2, the most effective as sEH and hyaluronidase (Figure 5D–5F). sEH is flavonoids, is one of most hydrophobic flavonoids known as a pro-inflammatory factor by metabolizing in this study. But, the relationship between anti-inflammatory epoxyeicosatrienoic acids (EETs) hydrophobicity of flavonoids and inhibition activities to less potent dihydroxyeicosatrienoic acids (DHETs) [29]. Relationship of inflammation to hyaluronidase is considered not so high, because the inhibition [30] activities of other most hydrophobic flavonoids, such is also reported . Therefore, naringenin is thought as FLV1 and FLV4, were not so high compare to to work as an enzyme inhibitor, too. The wide other tested flavonoids. difference of effective concentration of naringenin

12 Azuma S, et al. was observed on each inflammatory related Citrus unshiu Markovich (1). Yakugaku Zasshi 1989; parameter (Figure 5), but this phenomenon is also 109(11): 835–842. observed in case of other flavonoids[31]. 2. Matsuda H, Yano M, Kubo M, et al. Iinuma M, Oyama M, Mizuno M. Pharmacological study on In this study, we clarified the large difference citrus fruits. II. Anti-allergic effect of fruit of Citrus between narirutin and its aglycone naringenin unshiu MARKOVICH (2). On flavonoid components. on anti-inflammatory effects, and suggested the Yakugaku Zasshi 1991; 111(3): 193–198. importance of deglycosylation of narirutin to 3. Nishiura H, Yasui T, Tanaka K. Cosmetic effect of naringenin at oral ingestion of C. jabara fruit peels. Citrus jabara peel extract. Fragrance J 2007; 35: 86–88. In fact, when patients with allergic disease intake C. 4. Minatoguchi K, Oono Y, Funaguchi N. Effect of jabara fruit peels, there are individual differences “Jabara” juice on symptoms and QOL in patients with on anti-allergic and anti-inflammatory effects. Our Japanese cedar pollinosis. Clin Immunol Allergol findings may contribute to dissolve this problem. 2008–2009; 50: 360–364. As shown in Figure 1, narirutin is a flavanone that 5. Iwashita J, Iguchi N, Takashima A, et al. Citrus contains rutinose group (Glc-Rham). Although jabara extracts suppress MUC5AC mucin production hydrolysis of rhamnosides was not observed in in human lung epithelial cells. Adv Biol Chem 2017; human small intestinal cell extract[32], but rhamnosyl 7: 139–150. doi: 10.4236/abc.2017.73009. moiety of flavonoids could be hydrolyzed by strains 6. Abkenar A, Isshiki S. Molecular characterization of intestinal Bacteroides from humans[33]. Therefore, and genetic diversity among Japanese acid intestinal bacterial flora is considered as an important citrus (Citrus spp.) based on RAPD markers. J Hortic Sci Biotech 2003; 78: 108–112. doi: element for C. jabara fruit peels to exhibit anti- 10.1080/14620316.2003.11511574. inflammatory effects. Now, we are trying to explore 7. Penjor T, Yamamoto M, Uehara M, et al. most suitable intestinal bacteria for deglycosylation Phylogenetic relationships of Citrus and its relatives of narirutin in C. jabara fruit peels in order to based on matK gene sequences. PLoS One 2013; be more effective in more patients with allergic 8(4): e62574. doi: 10.1371/journal.pone.0062574. diseases. 8. Shimizu T, Kitajima A, Nonaka K, et al. Hybrid origins of citrus varieties inferred from DNA marker Conclusion analysis of nuclear and organelle genomes. PLoS One 2016; 11(11): e0166969. doi: 10.1371/journal. C. jabara was a unique citrus that almost all of pone.0166969. flavonoids in fruit peels were narirutin. Although 9. Chen HJ, Chung CP, Chiang W. Anti-inflammatory anti-inflammatory effects of narirutin was weak, effects and chemical study of a flavonoid-enriched but, its aglycone naringenin exhibited following fraction from adlay bran. Food Chem 2011; 126(4): inhibitory effects: NO synthesis, iNOS induction, 1741–1748. doi: 10.1016/j.foodchem.2010.12.074. IL-6 synthesis, and sEH activity. Since narirutin is 10. Sugiyama S, Umehara K, Kuroyanagi M, et al. deglycosylated to naringenin that is then absorbed Studies on the differentiation inducers of myeloid leukemic cells from Citrus species. Chem Pharm by colonocytes, it is considered that narirutin exists Bull (Tokyo) 1993; 41(4): 714–719. doi: 10.1248/ like a prodrug and its aglycone naringenin works as cpb.41.714. an active form of anti-inflammatory effect in a living 11. Lichius JJ, Thoison O, Montagnac A, et al. body at oral ingestion of C. jabara fruit peels. Antimitotic and cytotoxic flavonols from Zieridium pseudobtusifolium and Acronychia porter. J Nat Prod Acknowledgements 1994; 57(7): 1012–1016. doi: 10.1021/np50109a024. 12. Akachi T, Shiina Y, Ohishi Y, et al. Hepatoprotective This study was partially supported by the project effects of flavonoids from Shekwasha (Citrus “Research and development support project for depressa) against D-galactosamine-induced liver pioneering industrial technology by Wakayama injury in rats. J Nutr Sci Vitaminol (Tokyo) 2010; Prefectural Government”. 56(1): 60–67. doi: 10.3177/jnsv.56.60. 13. Li S, Lo CY, Ho HT. Hydroxylated Poly Conflict of interest methoxyflavones and Methylated Flavonoids in Sweet Orange (Citrus sinensis) Peel. J Agric Food The authors declare no potential conflicts of Chem 2006; 54(12): 4176–4185. doi: 10.1021/ interest. jf060234n. 14. Matsubara R, Yamada H, Ju-ichi M, et al. Polymethoxyflavonoids from the peel of Citrus References jabara. Shoyakugaku Zasshi 2012; 66(1): 23–25. 15. Tosa S, Ishihara S, Toyota M, et al. Studies of 1. Kubo M, Yano M, Matsuda H. Pharmacological flavonoid in Citrus. Analysis of flavanone glycosides study on citrus fruits. I. Anti-allergic effect of fruit of in the peel of Citrus by high performance liquid

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